We have compelling evidence in both wild-type (+/+) and estrogen receptor-alpha (ER-alpha) gene-disrupted (-/-) (ERKO) postnatal day- (P7) 7 mouse neocortex and uterus that points strongly to the existence of a novel, plasma-membrane-associated, putative ER that is neither ER-alpha (67 kDa) nor ER-beta (54-64 kDa). The proposed studies are designed to further characterize this putative ER, which I have designated "ER-X." "ER-X" is an about 62-63 kDa protein that: (a) is expressed and enriched in purified caveolar-like microdomains of P7, but not adult, neocortical and uterine plasma-membranes; (b) binds 3H-estradiol with high affinity but with ligand specificities radically different from ER-alpha and ER-beta; (c) exhibits some homology with the ligand binding domain of ER-alpha; (d) appears to mediate estrogen activation of the Mitogen-Activated Protein Kinase (MAPK) cascade; (e) is developmentally regulated; and (f) is up-regulated in adult neocortical tissue surrounding an ischemic stroke. We will use standard methods in a series of complementary and correlative cell biological, biochemical, and molecular biological experiments in developing wild-type and ERKO neocortex, using intact tissue; organotypic cultures; and plasma membrane preparations. These methods will include (i) competitive displacement assays, by comparison with 17betaestradiol, (ii) immunoprecipitation and Western blotting; (iii) in situ hybridization; (iv) immunohistochemistry; and (v) cDNA microarray analysis. The experiments are designed to determine (1) Whether "ER-X" is a multiligand receptor; a G-protein coupled receptor or a receptor tyrosine kinase; and (2) To characterize, quantify and compare the genes regulated by ER-X exposure to 17alpha- and 17beta-estradiol. These studies have profound implications for CNS development and cognitive disorders. The results will provide new information regarding the developmental actions and neuroprotective effects of estrogen. Understanding how estrogen influences development may help explain findings that estrogen exerts effects on higher order cognitive processes, and that estrogen or its deficiency may be risk factors for the development of the sexually dimorphic disorders of cognition; schizophrenia; Turner's and Down's Syndromes; and cognitive deficits associated with AIzheimer's and Parkinson's diseases. Understanding developmental mechanisms involving "ER-X" have considerable clinical significance for therapeutic approaches to cognitive deficits, leading the way to drug development and therapeutic intervention selective for "ER-X" in the brain.